General Histology & Molecular Techniques Flashcards

Question

Explain Principles of PCR

Answer 1

_PCR = polymerase chain reaction_ lab technique for rapid production or amplification of millions to billions of copies of a specific segment of DNA which can then be studied in more detail Involves using short synthetic DNA fragments called primers to select the segment of genome to be amplified Then multiple rounds of DNA synthesis to amplify that sequence _Need:_ DNA, primers, TAQ polymerase, nucleotides and buffer mix in a PCR tube and a thermal cycler _Steps:_ Denaturing, annealing and extension

Answer 2

For somatic mutations in BRAF, EGFR, KIT, KRAS, and NRAS genes The OncoFOCUS Panel employs two multiplexed PCR reactions. Amplification is followed by a single base extension reaction. The extension products are dispensed onto a Chip Array and detected via mass spectrometry. After the sample run, the OncoMutation Report provides the list of positive mutations identified in the samples.

Answer 3

Mass spectrometry (MS) is a technique used to measure the mass-to-charge ratio of ions. The results are presented as a mass spectrum, a plot of intensity as a function of the mass-to-charge ratio. _Three steps_: 1) Ionisation e.g. by bombarding substance with electrons to create positively charged fragments 2) Fragments then separated according to mass:charge ratio by exposing them to electomagnetic field 3) Ions then detected & sorted using an electron multiplier When a specific combination of source, analyzer, and detector becomes conventional in practice, a compound acronym may arise to designate it succinctly. One example is MALDI-TOF, which refers to a combination of a matrix-assisted laser desorption/ionization source with a time-of-flight mass analyzer. (Used with Oncofocus).

Answer 4

_Electron microscopy is useful for:_ Typing Glomerulonephritis. Metabolic Inherited Disorders (Heart / Liver) Peripheral neuropathy Myopathies Primary ciliary dyskinesia -\> Also rapid viral identification, help with tumour typing

Answer 5

1. Small 1mm cube of tissue 2. Primary fixation in glutaraldehyde 3. Secondary fixation in osmium tetroxide 4. Rinse and dehydration steps (ethanol) 5. Dry with liquid CO₂ 6. Embed by infusing with resin and cut thin (70nM) sections with diamond blade or cleaved glass 7. Post stain and image

Answer 6

Birbeck granules - Langerhan cells

Answer 7

Primary ciliary dyskinesia Circle and arrow = loss / reduction of dynein arms

Answer 8

Fabry disease! (dense lamellar bodies or Zebra bodies)

Answer 9

Leydig cell tumour with Reinke crystals

Answer 10

Juxtaglomerular cell tumour Rhomboid shaped pro-renin granules in cytoplasm

Answer 11

Malignant mesothelioma with microvilli (and tight junctions)

Answer 12

Neuroblastoma with dysmorphic synaptic vesicles containing neurosecretory granules

Answer 13

_MMR testing_ performed using immunohistochemistry looking for presence or absence of MMR proteins (MLH1, PMS, MSH2, MSH6) _MSI testing_ is performed by PCR looking at DNA for an increase in nucleotide repeats between tumour and patients normal tissue _Discordance?_ - check the same sample was used for both, not a mix up - low tumour volume in MSI sample (eg very mucinous) - if MSI testing is positive but IHC negative this may be due to a MMR protein tht is present but non functional

Answer 14

1. DNA is extracted from tumor areas on unstained slides and _treated with bisulfite_, which converts unmethylated cytosine residues to uracil. Methylated cytosines are protected from conversion. 2. Treated DNA is then amplified by _real-time PCR_. A 100 bp segment of the MLH1 promoter known to contain eight CpG dinucleotides and be subject to methylation is targeted. 3. _Temperature dissociation._ The amplified product is detected with intercalating dye. The melt characteristics of the generated amplicons are determined by loss of fluorescence as the double-stranded molecule is heated and becomes single-stranded. The temperature at which the amplicon dissociates is dependent upon the sequence, and indicates whether the sequence was methylated or unmethylated.

Answer 15

BRAF is a proto-oncogene within the MAPK pathway Following a mutation within the gene it becomes an oncogene and becomes constitutively active driving cell signalling pathways and cell proliferation BRAF mutations seen in wide range of cancers: PTC, LCH, ECG, HCL, melanoma, lung, CRC Presence of BRAF mutation means that BRAF inhibitor therapy may be an option for the patient Also if a BRAF mutation is present then it is unlikely that TKI's for upstream targets e.g EGFR will be effective

Answer 16

ERBB2 / HER2 / Neu is a proto-oncogene which codes for the HER2 receptor tyrosine kinase (member of EGFR family) Amplification or overexpression of the HER2 gene resulting in activation of multiple signalling pathways (MAPK, STAT, p13K) which promote cell proliferation and prevent apoptosis Implicated in multiple cancers: breast, gastric, salivary duct carcinoma, ovarian, lung adenocarcinomas Important molecular target for monoclonal antibodies such as trastuzumab (Herceptin)

Answer 17

EGFR is a receptor tyrosine kinase involved in cell signalling pathways that regulate cell division and survival Amplification and mutations in EGFR gene lead to a constitutively active tyrosine kinase receptor with sustained cell signalling and proliferation EGFR is implicated in a number of tumours (e.g lung adenocarcinoma and high grade gliomas) and is targetable Long term use of TKI's lead to drug resistance e.g. EGFRT790M - identifying this means patient can switch to 3rd generation TKI

Answer 18

RAS - family of genes and proteins (KRAS / NRAS / HRAS) involved in the MAPK signalling pathway Expressed in all cell lineages and mutations that activate RAS are found in up to 20% of all human cancers (CRC, pancreatic up to 90%, lung adenocarcinomas) Control cell signalling and cell proliferation If KRAS mutation is present then EGFR TKI's will be ineffective (as KRAS is downstream)

Answer 19

MYC is a proto-oncogene that codes for transcription factors If MYC rearranges (e.g MYC-IgH translocation in BL) or amplified (e.g. neuroblastoma, medulloblastoma) then transcription factors are produced at a higher rate

Answer 20

RB1 gene (13q) codes for the RB1 protein which is a negative regulator of the cell cycle RB1 protein prevents cell cycle progression from G1 to S phase Germline mutation = retinoblastoma (double hit) and osteosarcoma Somatic mutations = spindle cell lipoma as well as some common cancers (lung, PDAC, breast) Hijacked and disarmed by E7 oncoprotein following infection by HR HPV

Answer 21

VHL is a tumour suppressor gene which encodes a multifactorial protein that negatively regulates HIF Germline mutations = VHL syndrome Associated tumours: ccRCC, haemangioblastoma, phaeo, endolymphatic sac tumour, epididymal papillary cystadenoma, serous cystadenoma of pancrease, PanNET's with clear cells

Answer 22

APC is a tumour suppressor gene that codes for the APC protein which is a negative regulator of the cell cycle APC controls ß-catenin concentrations and interacts with e-cadherin as part of Wnt pathway Germline loss = FAP syndrome, attenuated FAP, Gardner, Turcot Somatic mutation = colorectal cancer

Answer 23

TP53 gene codes ofr p53 protein which induces cell cycle arrest, DNA repair and apoptosis. "guardian of the genome" - mutations of p53 implicated in 1/2 - 1/3 all human cancers Li Fraumeni - germline loss of one TP53 gene (high lifetime risk of breast, GBM, osteosarcoma, soft tissue sarcomas, adrenal cortical carcinoma) p53 hijacked by E6 oncoprotein in hr HPV infection

Answer 24

BRCA2 gene codes of BRCA2 protein which mediates the orderly assembly of DNA repair proteins Germline mutation: BRCA2 = hereditary breast and ovarian cancer Breast cancer, including male Ovarian cancer, especially serous Prostate Pancreas Sporadic: Breast cancer and NSCLC

Answer 25

CDKN2A is a gene ubiquitously expressed in many tissues and cells. The gene codes for two proteins p16 and p14arf. p16 and p14arf act as tumor suppressors by regulating the cell cycle. p16 activates the retinoblastoma (Rb) family of proteins, which block traversal from G1 to S-phase. p14ARF activates the p53 tumor suppressor Somatic mutations common in many cancers including melanoma, mesothelioma and high grade gliomas. Germline mutations of CDKN2A are associated with familial melanoma, glioblastoma and pancreatic cancer

Answer 26

NF1 gene codes for the neurofibromin protein, a GTPase activating protein that negatively regulates the RAS / MAPK pathway NF1 is the most common single gene disorder in humans. AD but half of cases arise sporadically, phenotypic variability NF1 - diffuse and plexifrom neurofibromas, multiple NF's, cafe au lait spots, Lisch nodules in iris, PA of optic nerve Sporadic mutations seen in GIST, melanoma, lung and GBM

Answer 27

Typical electropherogram covers a single PCR product (average # of nucleotides 200 - 600) depending on size of gene being sequenced Comparative analysis of patient sample and a DNA sample without a mutation Each peak represents a single nucleotide in the DNA sequence. Each nucleotide has a differnt colour (A = green, T=red. C=blue, G= black) Sequencing analysis usually performed by software

Answer 28

Molcular cytogenetic technique for _analysing copy number variations relative to ploidy level_ in the _DNA of a test sample compared with a reference sample_ Aim to compare 2 genomic DNA samples from 2 sources because they are suspected to have differences in gains or losses of chromosomes / subchromosomes Array CGH with competitive FISH has a resolution of 5 - 10 kilobases

Answer 29

Sequencing of multiple specimens in parallel Targeted gene panel, whole exome, or whole genome Multiple commercially available platforms with varying engineering configurations and forms of sequencing chemistry _Basic Steps:_ 1. Build library by extracting DNA and amplification by PCR 2. Sequencing 3. Data extraction and bioinformatics